The Role of Axl and Axl-like Proteins in Murine Spermatogenesis

Abstract

Mice lacking the Axl receptor tyrosine kinase (RTK) and its family members exhibit detrimental effects on their reproductive ability. AXL is localized to Sertoli cells, which are the major nurturing cells in the seminiferous epithelium. A sequence homology search identified an uncharacterized protein, AXL-LIKE, which we hypothesize is an endogenous dominant-negative of AXL. The structure of AXL-LIKE is essentially identical to AXL in the extracellular domain, but the intracellular portion of AXL-LIKE lacks the tyrosine kinase domain. The purpose of this study was to determine the expression of Axl-like within early developing testes, as well as its interaction with AXL and the role of AXL-LIKE in AXL function. Relative Axl and Axl-like mRNA transcript levels were highest in early postnatal testes, specifically at 7 days post partum (dpp), compared to 14dpp and 21dpp. Axl transcript levels on embryonic day 15 (e15) were significantly less than on 7dpp for Axl, but not Axl-like. Overexpression of AXL in COS-7 cells increased filopodial number, which was suppressed by co-expressing AXL-LIKE. Cells co-expressing AXL and AXL-LIKE were smaller and rounded compared to control or AXL only overexpressing cells. There was a redistribution of AXL localization, from cell membrane to vesicular bodies, when AXL-LIKE and AXL were co-expressed. Immunoprecipitation studies determined AXL and AXL-LIKE were not associated with one another; however introduction of AXL-LIKE reduced the amount of AXL present within cells as determined by western blot analysis. Overexpression of AXL increased cellular protein phosphorylation and this effect was eliminated when AXL-LIKE was co-expressed. The mRNA transcript levels detected in the testes and embryonic tissue using real time-PCR appear to be correlated with the number of Sertoli cells. This suggests a role for AXL-LIKE within this cell type. This study is the first to show that expression of AXL-LIKE, an endogenous truncated isoform of Axl, inhibits the function of AXL in vitro. Though no direct interaction was observed between the two proteins, AXL-LIKE may affect a downstream signaling pathway of AXL or cause alterations in the recycling of AXL.